This invention is in the fields of pharmacology and neurology. It relates to two different classes of receptors on the surfaces of neurons, known as NMDA receptors, which are triggered by N-methyl-D-aspartate (NMDA), and GABA receptors, which are triggered by gamma-aminobutyric acid (GABA).
This invention involves the use of NMDA antagonists (i.e., agents which block activity at NMDA receptors) as therapeutic agents which can prevent excitotoxic brain damage and nerve cell death during stroke, cardiac arrest, perinatal asphyxia, drowning, and various other events. Unfortunately, the currently available NMDA antagonists, when used for such purposes, exert toxic side effects that can kill or permanently damage neurons in certain regions of the brain.
This invention relates to the discovery that certain types of barbiturates which function as GABAmimetic agents (i.e., they trigger activity at GABA receptors) function as "safening agents" to reduce the damaging side effects of NMDA antagonists. By reducing the damage caused by NMDA antagonists, the barbiturates disclosed herein allow the safe use of NMDA antagonists to treat stroke, cardiac arrest, and other conditions.
To understand the processes and interactions involved, some background information is necessary on NMDA and GABA receptors, and on various types of molecules that stimulate or suppress activity at those receptors.
Receptors, messenger molecules, agonists, and antagonist
The surfaces of nerve cells in the central nervous system (the CNS, which includes the brain, spinal cord, and retina) contain various types of receptor molecules. In general, a receptor molecule is a polypeptide that straddles a cell membrane. When a messenger molecule interacts with the exposed extracellular portion of the membrane receptor molecule, it triggers a difference in the electrochemical status of the intracellular portion of the receptor, which in turn provokes some response by the cell. The messenger molecule does not bond to the receptor; instead, it usually disengages from the receptor after a brief period and returns to the extracellular fluid. Most receptor molecules are named according to the messenger molecules which bind to them.
An "agonist" is any molecule, including the naturally occurring messenger molecule, which can temporarily bind to and activate a certain type of receptor. An agonist can cause the same effect as the natural messenger molecule, or in some cases it can cause a more intense effect (for example, if it has a tighter affinity for the receptor molecule and remains bound to the receptor for a prolonged period). By contrast, an "antagonist" is a molecule which can block or reduce the effects exerted by the natural messenger molecule.
The role a specific molecule plays as an agonist or antagonist must be viewed with regard to a certain type of receptor. For example, while PCP and MK-801 are antagonists for the NMDA receptor, they are agonists for the PCP receptor (both types of receptors are discussed below). Most agonists and antagonists are xenobiotic drugs, i.e., they do not exist naturally in the body.
Membrane receptors involved in the transmission of nerve impulses between neurons are divided into two main categories: excitatory receptors, and inhibitory receptors. In general, excitatory receptors initiate or facilitate the conduction of nerve impulses. The principle class of excitatory receptors is referred to as "excitatory amino acid" (EAA) receptors, or as "glutamate" receptors. EAA receptors are important to this invention, and they are discussed below. A second major class of excitatory receptors, called cholinergic receptors, are activated by acetylcholine. They are discussed in the co-pending parent application, which issued as U.S. Pat. No. 5,034,400. That patent describes the use of anti-cholinergic drugs as safening agents to reduce the neurotoxic side effects of NMDA antagonists. Since the invention discussed below does not heavily involve cholinergic receptors or anti-cholinergic drugs, they are not discussed in detail herein.
In contrast to excitatory receptors, inhibitory receptors help to suppress the initiation or conduction of nerve impulses. Inhibitory receptors include GABA receptors, which are highly important in this invention, as well as other types of receptors that are not important herein, such as dopamine, serotonin, and opiate receptors. GABA receptors are discussed below, under their own heading.
For more information on neuroanatomy, neurotransmitters, receptors molecules, and agonists and antagonists which interact with CNS receptors, see Adelman 1987 (complete citations are provided below).
Excitatory amino acids (EAA's) and neurotoxicity
Two molecules that are highly important in the functioning of the CNS are glutamate and aspartate, which together are called excitatory amino acids (EAA's). Both molecules are found naturally in high concentrations in the central nervous system (CNS), where they function as excitatory neurotransmitters. Since glutamate is the predominant excitatory neurotransmitter, the following discussion will focus primarily on it.
Under normal conditions, when glutamate is released into a synaptic junction between two neurons, it reacts with and triggers an EAA receptor. This event is the key step in the process of neurotransmission. The glutamate is then transported back inside a neuron by means of a transport mechanism that requires energy. Glutamate is not allowed to accumulate in the synaptic fluid, since it would generate spurious and undesirable messages. However, under severe low energy conditions such as hypoxia/ischemia (as occurs in various conditions such as stroke), the transport system that normally transports glutamate back into neurons lacks sufficient energy to function properly.
For its energy needs, the brain is totally dependent on oxygen and glucose. Since the brain contains virtually no reserve supply of carbohydrates, it must rely on oxygen and glucose supplied by the blood. Hypoxia refers to a state of inadequate oxygen, and ischemia refers to inadequate blood supply (which directly entails a reduced supply of oxygen and glucose). Hypoxia and ischemia are encountered in various conditions such as stroke, cardiac arrest, loss of blood due to an injury, anemia, carbon monoxide poisoning, drowning, suffocation, or perinatal asphyxia.
When energy deficiency associated with hypoxia or ischemia impairs the glutamate transport system, glutamate accumulates in synaptic junctions and excessively stimulates EAA receptors. When a receptor-bearing neuron is excessively excited by this process, it discharges its own glutamate onto EAA receptors on other neurons. This leads to an "excitotoxic" process involving a cascade of increasing glutamate release, as more and more neurons become over-stimulated and begin firing in an uncontrolled manner until they literally excite each other to death. This process can result in widespread cell death that extends well beyond the initially affected area, and if not interrupted, can result in the death of the animal or person.
As used herein, the word "excitotoxic" refers to the process by which excitatory amino acids (primarily glutamate and aspartate) kill neurons by means involving excessive excitation. Since neurons in the CNS are not regenerated after they die, excitotoxic cell death leads to permanent and irreversible brain damage even if the person or animal survives. As an example, babies that suffer perinatal asphyxia often go through their entire lives with severe and crippling cerebral palsy, even though the disruption of oxygen supply to their brains may have lasted for only a few minutes.
In addition to their role in brain damage associated with hypoxia and ischemia, glutamate and aspartate are also believed to be implicated in various other neurological disorders involving the death of neurons, including alcoholism, epilepsy, trauma of the brain or spinal cord, and slowly developing neurodegenerative disorders such as Huntington's, Parkinson's and Alzheimer's diseases.
Other mechanisms by which EAA's can cause neuronal injury include abnormal sensitivity of EAA receptors to the excitatory action of EAA's, and the presence of abnormal molecules (such as glutamate analogs, certain types of food poisons, etc.) with excitotoxic properties. In some cases, such receptor-triggering molecules can accumulate at EAA receptors because they are not recognized by the cellular transport systems as molecules which should be removed from the extracellular fluid.
Glutamate and asparate are sometimes called "endogenous" excitotoxins, meaning that they are naturally synthesized and maintained in significant concentrations within the CNS. By contrast, if glutamate or aspartate or their excitotoxic analogs are ingested in foods or administered systemically, they are referred to as "exogenous" excitotoxins.
For two review articles which summarize numerous reports on excitotoxicity, see Olney 1989 and Rothman and Olney 1986.
EAA receptors, also known as glutamate receptors, are categorized into three subtypes. Each receptor type is named after a glutamate analog that selectively excites that particular class of receptor: N-methyl-D-aspartate (NMDA), kainic acid (KA), and quisqualate (QUIS). Glutamate is capable of activating all three receptor subtypes. Since NMDA receptors are the predominant type, KA and QUIS receptors are often grouped together and called non-NMDA receptors. QUIS receptors are sometimes called AMPA receptors, because AMPA was recently shown to trigger these receptors with greater specificity than quisqualate.
In the various neurotoxic situations, a major method of preventing or minimizing excitotoxic injury to the neurons involves administering drugs that selectively block or antagonize the action of the excitotoxic molecules at the EAA receptors.
NMDA Antagonists: MK-801, PCP, etc.
The EAA receptor subtype that has been implicated most frequently in neurodegenerative diseases and neurotoxicity is the NMDA receptor. An entire issue of Trends in Neurosciences (Vol. 10, Issue 7, July 1987) was devoted to review articles pertaining to the NMDA receptor, and to NMDA "antagonists" (i.e., molecules which can block or reduce the effects of NMDA agonists, including glutamate, at NMDA receptors).
Agents which act by binding directly to NMDA receptors, such as D-2-amino-5-phosphonopropanoate (D-AP5) and D-2-amino-7-phosphonoheptanoate (D-AP7), are referred to as "competitive" NMDA antagonists. Those two compounds are of limited therapeutic utility because they do not readily penetrate the blood-brain barrier. However, some competitive NMDA antagonists, including 2-amino-4-methyl-5-phosphono-3-pentenoic acid (common name CGP 37849), 4-phosphonomethyl)-2-piperidinecarboxylic acid (common name CGS 19755; Boast 1988) and 3-(2)-carboxypiperazine-4-yl)-propyl-1-phosphonate (common name CPP) and its unsaturated analog, CPP-ene (Herrling et al 1989 and Aebischer et al 1989) appear to permeate mammalian BBB's in sufficient quantity to affect the CNS after administration of relatively large quantities.
The NMDA receptor complex is also believed to contain at least two other binding sites, the glycine binding site and the polyamine binding site (reviewed in Olney 1989). Several drugs reportedly block activity at NMDA receptors by binding to those sites, and accordingly are regarded as non-competitive NMDA antagonists. Two drugs which reportedly bind to the polyamine site include 4-benzyl-alpha-(p-hydroxy-phenyl)-beta-methyl-1-piperidine-ethanol (commonly called ifenprodil) and (.+-.)-2-(4-chlorophenyl)-4-[(4-fluorophenyl)methyl]-1-piperidine ethanol (commonly called SL-82.0715; Carter et al 1988 and 1989). Certain halogenated analogs of kynurenic acid, such as 7-chloro-kynurenate, are believed to bind to the glycine binding site, as well as to KA and QUIS (non-NMDA) receptors.
The most powerful and effective NMDA antagonists known at the present time act at another receptor, the phencyclidine (PCP) receptor, which is considered a component of an ion channel complex coupled to the NMDA receptor (Kemp et al 1987). These compounds are called "non-competitive" NMDA antagonists because they do not compete for binding sites at NMDA receptors. When phencyclidine or its analogs activate the PCP receptor, the flow of ions through the NMDA ion channel is blocked, so that when the NMDA receptor is activated by an EAA, the NMDA receptor response does not result in the flow of ion currents. This prevents the excitation of the neuron.
Four compounds which can activate the PCP receptor, and which therefore serve as non-competitive NMDA antagonists, are phencyclidine, MK-801, ketamine, tiletamine, and dextromethorphan. Each is discussed in more detail below. All of these agents can penetrate the blood-brain barrier.
Phencyclidine (PCP) was originally introduced into clinical medicine some 30 years ago as an anesthetic (Goodman and Gilman 1975). Shortly thereafter, it was withdrawn from the market because it was found to have hallucinogenic properties that invited illegal use by drug abusers.
MK-801 is a phencyclidine analog manufactured by Merck, Sharp and Dohme (Rahway, N.J.). The chemical name is 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine. MK-801 is discussed in U.S. Pat. Nos. 4,064,139 (Anderson et al 1977), 4,374,838 (Anderson et al 1983), 4,399,141 (Anderson et al 1983), and 4,888,347 (Woodruff et al 1989). The maleate salt of MK-801 is commonly called dizocilpine, and is available for animal research. Although MK-801 was briefly tested as an anticonvulsant in human clinical trials, it was soon withdrawn from further testing on humans, with no published explanation. More recently, MK-801 underwent preliminary testing in human trials in England as a neuroprotective agent against stroke. Again, it was withdrawn from testing with no published explanation. Subsequently, after the publication of a report (Olney et al 1989; also see Allen and Iversen 1990 and the accompanying response by Olney) which documented neuronal damage in the cingulate and retrosplenial cortices caused by MK-801, phencyclidine, ketamine, and tiletamine, the U.S. Food and Drug Administration effectively declared a moratorium on any testing of NMDA antagonists in humans. Despite its side effects, MK-801 continues to generate great interest among neurology researchers, since it is the most powerful NMDA antagonist known and since it is highly selective for the PCP receptor.
Ketamine, a drug manufactured by Parke Davis and marketed under the trade name Ketalar, is another non-competitive NMDA antagonist which activates PCP receptors (Kemp et al 1987). It is widely used in human and veterinary medicine as an anesthetic, largely because it is rapidly cleared from the system and has relatively brief, non-lingering effects and because it does not compromise cardiorespiratory functions. However, in humans, as the anesthesia wears off and the patient awakens, symptoms such as unpleasant dreams or visions, excitement, and occasionally irrational behavior occur in some patients (Physicians Desk Reference, 1990, p. 1616). Such symptoms are often referred to as a "ketamine emergence reaction." Currently, the widely accepted treatment to suppress these symptoms involves the administration of minor tranquilizers such as diazepam, a benzodiazepine drug that is widely sold under the trademark "Valium". In 1989 it was reported that ketamine, when administered at relatively high doses, can cause permanent neuronal damage in the cingulate and retrosplenial cortex regions of the brain, in a manner comparable to PCP and MK-801 (Olney et al 1989).
Tiletamine is a drug manufactured by A. H. Robins. It is currently used in veterinary medicine, and is widely used for anesthesia on house pets. Like PCP, MK-801 and ketamine, tiletamine is known to activate PCP receptors and is recognized as a non-competitive antagonist of the NMDA receptor-ion channel complex. Tiletamine was also tested and shown to cause neuronal damage in the cingulate and retrosplenial cortices, as reported in Olney et al 1989.
Dextromethorphan is also known to bind to PCP receptors, but it binds more weakly than any of the other four agents listed above, and it was not tested in the experiments discussed in Olney et al 1989. It also binds to a receptor known as the sigma opiate receptor, which is similar in some respects to the PCP receptor. Dextromethorphan is an ingredient in various cough syrups, some of which are illicitly abused by teenage drug users.
Because of its great potency and the ease with which it penetrates blood brain barriers, MK-801 has become the drug used most widely in animal experiments aimed at testing the neuroprotective properties of NMDA antagonists. Since it has been shown to protect CNS neurons against numerous types of degenerative processes including hypoxia/ischemia, prolonged seizures, hypoglycemia, thiamine deficiency, and head or spinal cord trauma, there is great interest in using MK-801 or other NMDA antagonists to prevent or minimize brain damage in humans.
However, the potential therapeutic uses of NMDA antagonists must be viewed with caution, because such agents can inflict their own type of neurological damage, as discussed below. The subject invention involves a class of "safening" agents that can be administered along with NMDA antagonists, to reduce or eliminate the dangers and deleterious side effects of NMDA antagonists without interfering with their beneficial effects.
The term "safening agent" is used herein to refer to a chemical that can reduce one or more adverse effects of another chemical. This term is borrowed from other fields of chemistry such as agricultural chemistry, where safening agents are applied to crop seeds or plants to give crops a high level of resistance to a herbicide. This allows the herbicide to be used at a concentration that is highly effective against weeds, while minimizing the adverse effects of the herbicide on the crop plants.
In an analogous manner, anticholinergic drugs (described in U.S. Pat. No. 5,034,400) and certain types of barbiturates (described herein) can be used as safening agents in conjunction with NMDA antagonists. These safening agents protect the CNS against adverse side effects (discussed below) caused by NMDA antagonists. This allows NMDA antagonists to be used safely and effectively as beneficial neuroprotective agents.
Neurotoxic side effects of NMDA antagonists
One of the potentially serious side effects of MK-801, phencyclidine, ketamine, and tiletamine is that they have been shown to damage certain types of neurons. In a series of experiments, MK-801, phencyclidine, and ketamine were given to adult rats to test for neuroprotection against seizure-related brain damage (Clifford et al 1989). Those NMDA antagonists did protect neurons in certain brain regions; however, they also caused adverse reactions in two highly important regions of the brain, the posterior cingulate cortex and the retrosplenial cerebral cortex (Olney et al 1989).
One neurotoxic reaction, which was observed during microscopic analysis of CNS tissue after the rats were sacrificed, consisted of the formation of vacuoles (membrane-enclosed spaces in the cytoplasm that are not present in normal cells) and the dissolution of mitochondria (energy-producing organelles inside the cells). Although these changes appeared to be reversible if the doses of MK-801 or phencyclidine were sufficiently low, it was subsequently discovered that irreversible necrosis of cingulate cortical neurons followed the administration of 5 mg/kg MK-801. In adult rats, the ED.sub.50 for producing vacuoles in cingulate neurons by MK-801 administration (i.e., the dosage of MK-801 which will produce vacuoles in 50% of the animals treated) is 0.18 mg/kg, injected intraperitoneally (Olney et al 1989). Since the doses of MK-801 used in animal experiments for protecting neurons against ischemic brain damage usually are in the range of 1 to 10 mg/kg, it appears that the use of MK-801 for therapeutic neuroprotection poses a major risk of inducing potentially serious neurotoxic side effects.
A second indication of neuronal injury induced by NMDA antagonists involves the appearance of proteins called "heat shock protein" (HSP) in the cingulate and retrosplenial cortical neurons that are vulnerable to the toxic vacuole reaction. Heat shock proteins are often expressed in cells that are subject to severe stress (Currie and White 1981). They were first noticed in cells that were immersed in water that was nearly but not quite hot enough to kill the cells, which explains the name "heat shock". Two major types of heat shock proteins have been detected in mammals, one having a molecular weight of about 72 kilodaltons, the other having a molecular weight of about 90 kd. The 72 kd type (which presumably varies somewhat between different mammalian species) has been detected in the brains of people who died of Alzheimer's disease. Sharp et al 1990 reported that a 72 kd HSP was expressed in the brains of rats treated with MK-801. Based on that abstract, the Inventor used an antibody binding technique to confirm that 72 kd HSP's were expressed in cingulate and restrosplenial cortical neurons in treated animals; the HSP response remained detectable over a two week period.
The Inventor has shown that the pathomorphological side effects (vacuoles and mitochondrial dissolution) and the HSP response caused by NMDA antagonists can both be prevented by anticholinergic agents. This suggests that the two pathological reactions may have a common mechanism, and the finding that anticholinergic agents can protect against the pathomorphological and HSP side effects of NMDA antagonists is of considerable importance in a therapeutic context, since it permits NMDA antagonists to be used with improved safety as neuroprotective drugs. It also indicates that the mechanism of NMDA antagonist neurotoxicity involves both the blockade of the NMDA class of EAA receptors, as well as activation of the M-1 subtype of muscarinic cholinergic receptor (the order of potencies of anticholinergic drugs for preventing NMDA antagonist neurotoxicity parallels the order of binding affinities of those drugs to M-1 receptors).
As mentioned previously, MK-801 and phencyclidine are non-competitive NMDA antagonists; they bind to the PCP receptor, which is part of the NMDA ion channel complex, but they do not bind directly to the NMDA receptor itself. The Inventor has also studied the question of whether competitive NMDA antagonists (agents that bind directly to the NMDA receptor protein) can also cause pathomorphological effects comparable to the adverse effects of non-competitive NMDA antagonists, and the evidence indicates that competitive NMDA antagonists cause the same type of neurotoxic side effects. For example, microinjection of a competitive NMDA antagonist (D-AP5), which normally does not penetrate the BBB, into the cingulate cortical region (Labruyere et al 1989) caused the same type of vacuole reaction induced by MK-801 or PCP. Similarly, intravenous injection of CPP, a competitive NMDA antagonist that does penetrate blood-brain barriers to a significant extent, also causes the same vacuole reaction that is induced by PCP or MK-801. These results suggest that any antagonist which blocks NMDA receptor ion channel functioning by any mechanism is likely to cause adverse side effects. An important implication of this finding is that recently developed competitive NMDA antagonists which may be able to penetrate the blood-brain barrier (BBB) in sufficient concentration to be used as neuroprotective drugs, such as CGS 19755, CPP, and CPP-ene, might not provide a safe alternative to the non-competitive NMDA antagonists, unless anti-cholinergic drugs or the barbiturates disclosed herein are used as safening agents.
Non-NMDA Receptors and Broad-Spectrum EAA Antagonists
As mentioned previously, KA and QUIS (AMPA) receptors are often grouped together and called non-NMDA receptors. They have a higher degree of cross-reactivity with each other than either type has with NMDA receptors; most compounds that activate KA receptors also have some degree of affinity for QUIS receptors, and vice-versa. As used herein, "non-NMDA antagonist" refers to any compound that can suppress activity at either KA or QUIS receptors, or both.
Several compounds have been shown to have broad-spectrum activity in blocking all types of EAA receptors (i.e., both NMDA and non-NMDA receptors). Such compounds include kynurenic acid and its halogenated analogs (such as 7-chlorokynurenate), and certain types of thiobarbiturates (especially thiamylal).
Another class of compounds called quinoxalinediones has been discovered to have a high degree of antagonistic activity at non-NMDA receptors (Honore et al 1988). Some of these compounds also display blocking activity at NMDA receptors. Although some types of quinoxalinedione (including 6-cyano-7-nitro-quinoxaline-2,3-dione (common name CNQX) and 6,7-dinitro-quinoxaline-2,3-dione (common name DNQX) do not appear to be able to penetrate blood-brain barriers in quantities sufficient to make them effective inside the CNS, a third type of quinoxalinedione reported in 1989, 6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione (common name NBQX, also designated as FG 9202) penetrated BBB's in sufficient quantities to have a significant effect inside the CNS (Honore et al 1989 and Sheardown et al 1989).
The Inventor has demonstrated that blockage of both NMDA and non-NMDA receptors by a mixture of MK-801 and a quinoxalinedione provided better and more effective protection against hypoxic/ischemic (excitotoxic) damage to the CNS than either agent could provide by itself. That discovery is described in co-pending U.S. patent application Ser. No. 467,139, the contents of which are hereby incorporated by reference.
The GABA Receptor Complex
The GABA.sub.A receptor complex (also called the GABA-benzodiazepine receptor) is a complicated multi-component entity, reviewed in Zorumski and Isenberg 1991. It is believed to contain three or more interrelated binding sites.
One of the receptor binding sites is triggered by GABA (gamma-amino-butyric acid), an inhibitory neurotransmitter that is synthesized inside certain neurons. After release into a synaptic junction, GABA reacts with the GABA receptor protein, activating the receptor and triggering the opening of a chloride ion channel. The opening of the chloride channel allows chloride ions to enter the neuron. This results in a polarization of the neuronal membrane in the synaptic region, which temporarily inhibits the ability of the neuron to conduct bioelectric impulses. After activating its receptor, the GABA molecule is rapidly transported back into the cell and/or degraded by an enzyme called GABA transaminase.
A second binding site in the GABA receptor complex is called the benzodiazepine receptor. As indicated by the name, it is activated by benzodiazepine drugs, such as diazepam (Valium). When the benzodiazepine binding site is activated, it increases the flow of chloride ions through the GABA complex ion channel. By potentiating (increasing) the nerve signal suppression effects of GABA, benzodiazepine drugs such as Valium function as sedatives, "anxiolytic" (anxiety-suppressing) and anti-convulsant agents. Benzodiazepines can potentiate (strengthen) the chloride channel opening properties of GABA, but they cannot open the chloride channels by themselves in the absence of GABA. Therefore, they are said to possess "indirect" GABA agonist activity. By contrast, barbiturates act as "direct" GABA agonists, as discussed below.
A third protein receptor site in the GABA receptor complex, called the t-butylbicyclophosphorothionate binding site, is believed to interact with barbiturate drugs such as pentobarbital, secobarbital and thiamylal. Although the mechanism involved at that binding site is poorly understood, activation of that receptor is associated with the sedative and possibly the anti-convulsive effects of barbiturates. As mentioned previously, at least some barbiturates are capable of acting as "direct" agonists at the GABA receptor; they can cause the chloride channel of a GABA receptor to open, even in the absence of GABA. This has been demonstrated by "voltage clamp" experiments using cultured neurons, discussed in various articles such as MacDonald and Barker 1978 and Akaike et al 1987.
A fourth binding site, called the gamma-butyrolactone binding site, has also been suggested. When activated by alpha-substituted gamma-butyrolactones, it apparently increases the effects of GABA.
GABA.sub.A receptor complexes are also believed to be involved in reactions involving other psychoactive agents, including alcohol and steroid anesthetics. It should also be noted that if any of the GABA.sub.A receptor sites are blocked by agents such as bicuculline or picrotoxin, adverse responses such as anxiogenesis or convulsions can result.
An entirely different class of GABA receptor complexes are activated by an anti-spasticity drug, baclofen. Those receptor complexes are designated as GABA.sub.B receptors. They appear to be insensitive to benzodiazepines and barbiturates. Since GABA.sub.A complexes are believed to be the major sites of action for the drugs involved in this invention, GABA.sub.B complexes will not be discussed in any detail herein. Any references herein to GABA sites, or to GABAergic or GABAmimetic agents, refer to GABA.sub.A receptor complexes and to agents that interact with such receptors.
Drugs that act at the GABA receptor complex (at any of the binding sites) and mimic or increase the inhibitory effects of GABA are often called "GABAmimetic" agents. A related term, "GABAergic," is usually used to refer to components of the GABA transmitter system, such as GABAergic neurons, axons, or synapses.
It is not entirely clear how activity at the benzodiazepine, butylbicyclophosphorothionate, or butyrolactone binding sites potentiate the effects of GABA, and various mechanisms may be involved. Some drugs are believed to prolong the amount of time that a molecule of GABA remains attached to the GABA receptor. Other agents may function by means such as enlarging the diameter of the ion channel, or by increasing the amount of time that the chloride channel remains open.
GABA receptor complexes are important to the subject invention because the Inventor has found that certain GABAmimetic barbiturates can decrease both the pathomorphological and the HSP effects of NMDA antagonists in vulnerable neurons. Of particular note is the fact that GABAmimetic barbiturates completely block the neurotoxic side effects of MK-801, a powerful NMDA antagonist, whereas benzodiazepines such as diazepam (Valium) provide only partial protection.
Barbiturates
Following common usage, "barbiturate" is used herein to include analogs of barbituric acid which have sedative-hypnotic (anesthetic) or anticonvulsant effects in mammals. Barbiturates have the following general structure: ##STR1## where X is oxygen (in the case of most barbiturates, such as secobarbital or pentobarbital) or sulfur (in the case of thiobarbiturates such as thiamylal), and where R.sub.1 through R.sub.4 are hydrogen atoms or organic groups.
Barbiturates have long been used for anesthetic purposes. However, at anesthetizing dosages, they frequently interfere with respiration, so they must be used in conjunction with mechanical respiratory devices, often called ventilators.
Most barbiturates are referred to as "anesthetic barbiturates." This distinguishes them from a separate class of non-anesthetic barbiturates such as phenobarbital, an anticonvulsant that does not have the same type of anesthetic effects that most barbiturates have. For more information on the distinctions between anesthetic and anticonvulsant barbiturates, see MacDonald and Barker 1978.
As mentioned above, some types of barbiturates have been shown in in vitro tests to be "direct" agonists at the GABA receptor, in the sense that they can cause the opening of the chloride channel of a GABA receptor complex even in the absence of GABA. Although not all barbiturates have been tested for this property, to the best of the Inventor's knowledge, all of the barbiturates which have demonstrable potency as direct GABA agonists have been anesthetic barbiturates, while anticonvulsant barbiturates such as phenobarbital lack potency as either direct GABA agonists or as anesthetics. Accordingly, there appears to be a correlation between anesthetic activity and direct GABA agonist activity. In the absence of evidence to the contrary, a barbiturate that falls within the anesthetic category can be presumed to be a direct GABA agonist.
It has been shown that some anesthetic barbiturates also function as EAA antagonists; i.e., they can block the excitatory action of glutamate at EAA receptors. U.S. Pat. No. 4,833,148 (Olney 1989) discloses the use of thiobarbiturates as EAA antagonists to reduce neurotoxic damage due to hypoxia and ischemia, as evidenced by the ability of barbiturates to protect chick retina tissue in vitro against neurotoxicity caused by addition of NMDA, kainic acid, quisqualic acid, or glutamate to the tissue culture liquid or by ischemia which is simulated by the removal of oxygen and glucose from the culture medium. The apparent basis for the efficacy of barbiturates in conferring such protection is that they block all EAA receptors (both NMDA and non-NMDA); under ischemic conditions, as simulated in the retina tissue tests, the excitotoxic process is triggered by glutamate acting at all EAA receptor types. Thiamylal was shown to be more effective than the other barbiturates tested in protecting against glutamate toxicity or simulated ischemic damage. This is consistent with the finding that thiamylal was more potent than other barbiturates in blocking both NMDA and non-NMDA receptors.
Neither barbiturates nor diazepam have been used with NMDA antagonists for the purpose of preventing pathomorphological neurotoxic side effects of NMDA antagonists. However, there is a long tradition of using diazepam (Valium) or other benzodiazepines together with ketamine in human anesthesia. Benzodiazepines (particularly diazepam) are considered the agents of choice by anesthesiologists for suppressing the psychotomimetic side effects of ketamine. This practice is based on a series of studies over the past 15 years in which benzodiazepines have been shown to be moderately effective in reducing the incidence of emergence reactions that occur in some patients anesthetized with ketamine. In most studies, benzodiazepines have been reported to moderately reduce the severity or incidence of emergence reactions rather than totally prevent such reactions.
In the voluminous literature pertaining to use of benzodiazepines for suppressing ketamine-induced emergence reactions, including recent review articles, no mention is made of any studies in which barbiturates were tested for this purpose. However, a computerized literature search revealed that in 1974, two doctors in Nigeria reported that a thiobarbiturate, thiopentone (called thiopental in the United States) was effective in reducing ketamine-induced emergence reactions in a group of 50 women who were undergoing minor gynecological surgery (Magbagbeola and Thomas 1974). Although Magbagbeola and Thomas stated that no respiratory problems were encountered at the relatively low doses of thiopental they used (between 2 and 3 mg/kg), respiratory interference by barbiturates is a major concern whenever barbiturates are used; by contrast, diazepam provides a method of suppressing ketamine emergence reactions without raising any questions or concerns regarding respiration. Neither the Nigerian doctors nor anyone else ever followed up the 1974 report with additional reports containing more evidence for the efficacy of barbiturates in suppressing emergence reactions when using ketamine for anesthetic purposes, and there is no indication in the literature that anesthesiologists currently use barbiturates for this purpose.
It should also be noted that the Nigerian report involved the use of 2 to 3 mg ketamine per kilogram of patient body weight, to anesthetize or sedate female patients for "minor gynecological operations." The 2 mg/kg dosage provoked the transient unpleasant sensations known as ketamine emergence reaction in a fraction (less than half) of the patients studied. By contrast, the minimum dosage which reliably caused vacuole formation in cingulate neurons in rats was more than 40 mg/kg, which is in the range of ketamine dosages used in rats to achieve anti-ischemic protection.
It should also be pointed out that, because of their activity as NMDA antagonists, barbiturates should be expected to produce the same adverse side effects as other NMDA antagonists, and to aggravate those side effects when co-administered with other NMDA antagonists. However, the Inventor has discovered that barbiturates do not produce such side effects; instead, they function as safening agents which reduce or eliminate the adverse side effects of NMDA antagonists, apparently because in the cingulate and retrosplenial cortex where the adverse side effects are seen, their strong agonist activity at GABA receptors overrides their antagonist activity at NMDA receptors.
The discovery that barbiturates which act as direct GABA agonists can reduce or prevent the neurotoxic side effects of NMDA antagonists is the essence of this invention. NMDA antagonists can be highly useful therapeutic agents in various neurological disorders, but suitable agents and methods are needed to reduce or prevent the adverse side effects of NMDA antagonists.
Therefore, one object of this invention is to provide effective safening agents that can be used in both human and veterinary medicine to reduce the neurotoxicity of NMDA antagonists such as MK-801 or PCP, thereby allowing NMDA antagonists to be used more safely as anesthetics, or as neuroprotective agents to prevent neuronal death due to stroke, cardiac arrest, perinatal asphyxia, and other conditions.
Another object of this invention is to disclose that certain barbiturates effectively and reliably prevent the pathomorphological and heat shock protein reactions caused by NMDA antagonists, which affect neurons in the cingulate and retrosplenial cortex.
Another object of this invention is to provide a mixture of an NMDA antagonist combined with a barbiturate safening agent. Such mixtures can be used to prevent or reduce damage to the CNS resulting from stroke, cardiac arrest, neonatal asphyxia, and numerous other neurological diseases, trauma, and degenerative processes.